Nucleic acid amplification can be used for sequencing, cloning, genetic mapping, and other forms of nucleic acid sequence amplification, or to determine an initial concentration of nucleic acid in a sample by constructing a standard curve of results from samples including known concentrations, or other forms of nucleic acid amplification. Nucleic acid amplification can be used for analyzing nucleic acids including, for example, DNA and RNA. Types of nucleic acid amplification include polymerase chain reaction (PCR), transcription mediated amplification (TMA), ligase chain reaction (LCR), strand-displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
In general, PCR relies on the ability of DNA-copying enzymes to remain stable at high temperatures. A single PCR cycle includes three major steps: denaturation, annealing, and extension. During the denaturation, a liquid sample is heated at approximately 94° C. During this process, double DNA strands “melt” open into single stranded DNA and all enzymatic reactions stop. During annealing, the single stranded DNA is cooled to 54° C. At this temperature, primers bind or “anneal” to the ends of the DNA strands. During extension, the sample is heated to 75° C. At this temperature, nucleotides add to the primers and eventually a complementary copy of the DNA template is formed. PCR analyses typically repeat this PCR cycle multiple times (e.g., about 40) to produce a large number of replicate DNA strands.
Real-time PCR can be used to detect a relative amount of nucleic acid present in a sample as the sample undergoes a plurality of PCR interrogation periods (e.g., cycles). For example, the sample may include markers that fluoresce when attached to double-stranded DNA. In this example, fluorescence detected by a detector is proportionate to the number of double-stranded DNA present in the sample. Thus, as PCR proceeds, fluorescence increases.
Transcription-associated amplification embodiments, e.g., nucleic acid sequence based amplification (NASBA) and transcription-mediated amplification (TMA), use substantially isothermal conditions and an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template by using a promoter-primer, a primer, an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates (dNTP), ribonucleoside triphosphates (rNTP), and a promoter-template complementary oligonucleotide, and optionally may also include other oligonucleotides. Assays may use detection probes for amplification of the nucleic acids in the reactions. During the amplification reaction, each detection probe binds to its specific amplicon and is converted to a form that produces a detectable signal (e.g., a hydrolysis-resistant chemiluminescent signal or higher fluorescence intensity than in the unconverted form).
A need continues to exist for analysis and accuracy in the data generated by nucleic acid amplification processes.